Despite the critical role of the thymus in T cell development, the mechanisms required to specify initial thymus fate during fetal development are not known. Understanding these mechanisms will facilitate the development of strategies to generate functional thymus organs in culture, and to rejuvenate the aged, involuted thymus. Thymus fate is specified in the ventral 3rd pharyngeal pouch (pp) endoderm between E9.5 and E11 during mouse fetal development; the dorsal domain of this pouch gives rise to the parathyroid glands. As the 3rd pp must be patterned into two distinct cell fates, mechanisms must be in place to ensure that both organs are properly specified. Previous work from our labs and others have implicated BMP2/4 and FGF8/10 signals in establishing thymus fate, while SHH signaling is required for parathyroid fate. These two signaling pathways appear to act in opposition; however, the mechanisms by which they interact with each other and regulate organ-specific transcriptional networks remain to be established. Recent collaborative experiments from the Richie and Manley labs suggest that the transcription factor TBX1 is a key regulator of the decision between thymus and parathyroid cell fate. We have shown that Tbx1 is expressed in the parathyroid domain of the 3rd pp endoderm, but not in the ventral thymus fated domain, and that ectopic TBX1 is sufficient to suppress Foxn1 expression. Based on our preliminary data we generated a testable model of thymus fate specification in which suggest that FGF8 and BMP4 are positive regulators, whereas SHH and Tbx1 antagonize thymus development. This project uses a novel approach to decipher the molecular network that specifies endodermal progenitors to a thymus fate, combining embryology, genetics, molecular biology, bioinformatics, and epigenetics. The results will provide a rational basis for programming early progenitors to generate a functional thymic epithelial cell network that supports thymopoiesis. We will test our model in the following Specific Aims. Aim 1. Test the hypothesis that multiple mechanisms suppress Tbx1 expression in the ventral domain of 3rd pp endoderm, and that this suppression is required to establish thymus fate. Aim 2. Test whether the ability of TBX1 to inhibit thymus differentiation is temporally restricted, and identify the mechanism by which ectopic TBX1 and/or SHH signaling blocks thymus fate. Aim 3. Test the hypothesis that progressive establishment of organ-specific epigenetic signatures determines thymus fate, and depends on cooperation between the BMP and FGF signaling pathways